These studies are designed in part to identify and characterize the crucial polypeptide substrates of viral transforming proteins which possess tyrosine protein kinase activity. Novel substrates will be sought by "Western Blotting" using anti-phosphotyrosine antibodies. Since there are indications that the phosphorylation of many of the substrates of the viral protein kinases does not contribute materially to cellular transformation, advantage will be taken of mutants of Rous sarcoma virus which encode a non- fatty acylated transforming protein. These mutant proteins posses undimished protein kinase activity but cannot transform cells. The small subset of the substrates of the wild-type protein which do not undergo phosphorylation in cells infected with these mutants will be identified and studied. In addition, a variety of immune reagents--tumor sera and sera produced by immunization with proteins expressed in bacteria-- will be prepared and used to proto-oncogenes. The properties and structure of these proteins will be determined and the cells in which they are expressed will be identified. The effect of growth factors on the protein kinase activity and the phosphorylation of these cellular proteins will be examined.